Two novel variants of uncertain significance in GP9 associated with Bernard-Soulier syndrome: Are they true mutations?

Bernard-Soulier syndrome (BSS) is an autosomal recessive major thrombocytopathy, the symptoms of which are mainly marked by mucocutaneous bleeding. This rare disease, initially described in the 1970s, is the result of an abnormal formation of the glycoprotein complex Ib-IX-V (GP Ib-IX-V), a platelet receptor of von Willebrand factor. A large number of mutations, sometimes involving the GP9 gene, have been described as possibly responsible for the disease. We report here the case of a BSS patient who presented with persistent thrombocytopenia (31x109/L) and decreased surface expression of GPIb-IX-V on large platelets with anisocytosis. Thorough molecular analyses disclosed two previously unreported GP9 variants, respectively c.230T>A (p.Leu77Gln) and c.255C>A (p.Asn85Lys). Both are likely to modify the conformation of GP-IX interactions with other glycoproteins of the Ib-IX-V complex and thus proper expression of this complex on the membrane of platelets.

Use of a thrombopoietin receptor agonist in von Willebrand disease type 2B (p.V1316M) with severe thrombocytopenia and intracranial hemorrhage.

We present here a 63-year old woman with a long history of immune thrombocytopenia. She was hospitalized for a traumatic intracranial hemorrhage with thrombocytopenia. Following inefficient treatment of four platelet transfusions, immunoglobulins, and corticosteroids, we initiated treatment with a thrombopoietin (TPO) receptor agonist (eltrombopag 25 mg/d) with a good efficacy. Her mother and sister also had chronic thrombocytopenia. Clinical history, hemostasis results, and gene analysis revealed von Willebrand disease (VWD) type 2B with the mutation (c.3946G>A; p.V1316M), which combines a von Willebrand factor defect with severe thrombocytopenia, as well as a thrombocytopathy. The efficacy of TPO receptor agonists appears to counterbalance, at least to some extent, the thrombocytopathy associated with this mutation. As such, the use of TPO receptor agonists could represent an alternative therapeutic approach in cases of VWD type 2B with severe thrombocytopenia.

Flow cytometry in hematological nonmalignant disorders.

Multiparameter flow cytometry (MFC) has become an integral part of the diagnosis and classification of hematological malignancies. However, several nonmalignant or premalignant disorders may benefit from this technology in hematology laboratories. This review provides information on the normal immunophenotypic characteristics of peripheral blood leukocyte subsets and their modifications in several clinical conditions. The usefulness of MFC and the specific markers that can be investigated in hyperlymphocytosis, infection, hypereosinophilia, paroxysmal nocturnal hemoglobinuria, and large granular lymphocyte disorders is described. Mention is also made of the developments of MFC for analyses of red blood cells or platelets.

[Thrombin generation test]. / Le test de génération de thrombine.

Thrombin is a key enzyme of the coagulation cascade, having both pro- and anticoagulant functions. Global haemostasis assay, the so-called thrombin generation test is appropriate for its assessment. Estimation of an individual's potential to generate thrombin may correlate more closely with a hyper- or hypo-coagulable phenotype, compared to traditional coagulation tests. In patients at risk of venous thrombosis, thrombin generation analysis may be utilized to detect underlying thrombophilia. In patients with documented venous thromboembolism, increased thrombin generation values are seen in those patients at greatest risk for recurrence. In patients with arterial vascular disease, the data are limited. In case of haemophilia thrombin generation assays reflect bleeding severity. It is applicable for monitoring of both conventional haemophilia treatment and inhibitor-bypassing therapy, which is needed when inhibitors develop in patients. Standardization of thrombin generation methods and determination of cut off-values are required before its application in clinical practice.

Platelet function analyser (PFA-100) results and von Willebrand factor deficiency: a 16-year 'real-world' experience.

The platelet function analyser (PFA-100) is a biological tool designed to explore primary haemostasis. This system has thus been widely demonstrated as reliable in detecting von Willebrand factor (VWF) deficiency. However, most studies were based on patients benefitting from regular medical care and accurate diagnosis, and it would seem probable that the results were somewhat optimistic, and do not reflect its performances in 'real-world' situations. We have chosen to study the reliability of PFA-100 for screening VWF ristocetin cofactor (VWF:RCo) deficiency. We retrospectively analysed the results (n = 6431) of 4027 patients referred to our centre between October 1997 and June 2013 and in whom PFA-Epi, PFA-ADP, and VWF:RCo activity had been evaluated. We studied the influence of blood group on the results and the performances of each method in a subgroup of 213 patients with genetically confirmed von Willebrand disease. We have shown that the PFA-100 system, in our experience, constitutes an excellent screening test for detecting VWF:RCo deficiency, whatever the clinical situation, in 'real-world' conditions. The negative predictive value (NPV), the positive predictive value, the sensitivity and the specificity were respectively: 0.98, 0.51, 0.98 and 0.40. When values adjusted for blood group are used, NPV and sensitivity are inferior to those using normal values which have not been adjusted for blood group. We have shown the PFA-100 method to be more efficient in screening for VWF deficiency than the VWF:RCo technique.

Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements.

Thirty per cent of patients with mild haemophilia A (MHA) present markedly different FVIII: C level when assayed by one-stage clotting and two-stage chromogenic assays. It is, therefore, a real clinical challenge to predict the individual bleeding risk of these patients. The aim of the present work was to study the relationship between the bleeding tendency of these patients with the results of a panel of phenotypic and genotypic tools. Thirty-six patients with MHA were included in this multicentre prospective clinical study. The severity of bleeding symptoms was evaluated using the ISTH/SSC score. FVIII:C levels were measured using an activated partial thromboplastin time-based one-stage FVIII assay (FVIII: C1) and three commercial chromogenic kits (FVIII:CR). FVIII antigen levels, thrombin generation measurement and FVIII gene mutation analysis were also performed. Our results showed that a one-stage FVIII: C assay cannot rule out the diagnosis of MHA, a combined use of FVIII:C1 with a FVIII:CR is suitable for detecting MHA. We observed that FVIII:CR results better reflected the clinical bleeding tendency of patients compared to FVIII:C1. We also observed a relationship between thrombin generation (TG) capacity and FVIII:CR of these patients. FVIII gene mutation analysis showed mutations previously reported in MHA patients with discrepant FVIII:C measurements, but with no predictive value of the individual bleeding phenotype of patients. Overall, we observed a relationship between chromogenic FVIII:C results, TG assay and bleeding tendency of patients with discrepant FVIII:C measurements, while FVIII:C1 was not well correlated with clinical bleeding phenotype in this particular population.

Management of bleeding in severe factor V deficiency with a factor V inhibitor.

Factor V (FV) inhibitor arises rarely after using fresh frozen plasma (FFP) to treat inherited FV deficiency and is often a real therapeutic challenge. Here, we report a patient with a severe FV deficiency who developed such an inhibitor and was then treated with recombinant activated FVII (rFVIIa) and platelet concentrates (PC). Monitoring was assessed by thrombin generation assay (TGA). PC were more effective than rFVIIa in treating bleeding, but there was no correlation between the TGA results and clinical efficacy.

[Acquired hemophilia A. A monocentric retrospective study of 39 patients]. / Hémophilie A acquise. Étude d'une série rétrospective monocentrique de 39 patients.

PURPOSE: Acquired haemophilia A (AHA) is a rare bleeding disorder, due to the presence of an inhibitor directed against factor VIII (FVIII). About 50% of the AHA are idiopathic, while the remaining 50% are related to an underlying disorder or condition (autoimmune diseases, malignancies, postpartum, etc.). PATIENTS AND METHODS: We report on a monocentric retrospective cohort of 39 patients with AHA. Data were collected and compared to recent published data. RESULTS: Thirty-nine patients were admitted for AHA between 1993 et 2011. Mean age at diagnosis was 71.3 years, and we noted a marked male predominance. Although the majority of patients presented a bleeding event at diagnosis (94.9%), the hemorrhagic mortality was low (2.6%). On the contrary, immunosuppressive morbidity and mortality were high in this elderly population. There was a clear correlation between initial FVIII inhibitor titer and complete remission delay. We did not identify prognostic factor for global survival. CONCLUSION: AHA is a rare but potentially fatal disorder. Rapidity of diagnosis and treatment initiation is crucial. Morbidity and mortality, particularly of infectious cause, due to immunosuppressive treatment, should lead to consider other available therapeutical options.

Prevalence, biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one-stage and chromogenic factor VIII activity.

BACKGROUND: In most laboratories, the severity of hemophilia A is assessed by the factor VIII activity (FVIII:C) one-stage assay. However, comparisons of these results with those of two-stage assays can reveal discrepancies and suggest misdiagnosis. PATIENTS/METHODS: In this monocentric study, we measured FVIII:C with two methods (one-stage chronometric and chromogenic assays) in 307 (173 families) patients with moderate/mild hemophilia A. To compare results, we used a chronometric/chromogenic ratio. Discrepancy was defined as a ratio < 0.5 or > 1.5. We studied their putative involvement at known FVIII functional sites, their interspecies conservation status, and their spatial position within the FVIII structure. RESULTS: Thirty-six patients from 17 families exhibited a discrepancy between the two assays: 12 (6.9%) families had a low ratio (< 0.5), and five (2.9%) families had a high ratio (> 1.5). Qualitative deficiency was diagnosed in about 16% of the families. Molecular studies were performed in 15 of these 17 families, resulting in each case in the identification of missense mutations, including three novel mutations. We were further able to propose a pathophysiologic explanation. CONCLUSIONS: In this monocentric study, we have demonstrated a discrepancy between FVIII:C assay results in 10% of families with moderate/mild hemophilia A. The prevalence of 'inverse' discrepancy (i.e. low chronometric/chromogenic ratio) is high as compared with previous reports. We suggest that both FVIII:C assays are recommended in patients with moderate/mild hemophilia A for a complete biological phenotype. This could also improve our knowledge of the FVIII structure-function relationships.

Hyperzincemia and hypercalprotectinemia: unsuccessful treatment with tacrolimus.

UNLABELLED: We report on a 5-year-old boy with hyperzincemia and hypercalprotectinemia. Treatment began with Tacrolimus at the age of 4 years and 6 months. Despite an initial correction of clinical and biological symptoms, zincemia and calprotectinemia progressively worsened with secondary reappearance of symptoms. CONCLUSION: Tacrolimus seems to have a transient effect in the treatment of Hyperzincemia and hyperprolactinemia.

Prospective evaluation of the '4Ts' score and particle gel immunoassay specific to heparin/PF4 for the diagnosis of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a severe disease that is often difficult to diagnose. A clinical scoring system, the '4Ts' score, has been proposed to estimate its probability before laboratory testing, and a particle gel immunoassay (H/PF4 PaGIA) has also been developed for rapid detection of HIT antibodies. AIM: To evaluate the performance of both methods when HIT is suspected clinically. METHODS: Two hundred thirteen consecutive patients were included in four centers. The probability of HIT was evaluated using the 4Ts score blind to antibody test results. HIT was confirmed only when the serotonin release assay (SRA) was positive. RESULTS: The risk of HIT was evaluated by the 4Ts score as low (LowR), intermediate (IR) or high (HR) in 34.7%, 60.6% and 4.7% of patients, respectively. The negative predictive value (NPV) of the 4Ts score was 100%, as the SRA was negative in all LowR patients. PaGIA was negative in 176 patients without HIT (99.4%, NPV) and the negative likelihood ratio (LR-) was 0.05. PaGIA was positive in 37 patients, including 21 with HIT (positive predictive value = 56.8%), with a positive LR of 11.4. A negative PaGIA result decreased the probability of HIT in IR patients from 10.9% before assay to 0.6%, whereas a positive result did not substantially increase the likelihood for HIT. CONCLUSION: The use of the 4Ts score with PaGIA appears to be a reliable strategy to rule out HIT.

CFU-Mk content of immunoselected CD34+ peripheral blood progenitor cells, evaluated with an adapted serum-free methylcellulose assay, is predictive of platelet lineage reconstitution in children with solid tumors.

Immunoselected CD34+ peripheral blood progenitor cell (PBPC) transplantation is now frequently used to support autologous hematopoiesis after myeloablative therapy, its feasability having been proved by several groups. However, we and others observed delayed platelet recovery. We hypothesized that immunoselection processing might induce selective loss of megakaryocyte progenitors, or a decrease in their proliferation. We used a colony-forming units megakaryocyte (CFU-Mk) assay to evaluate these consequences and predict platelet recovery in patients. In CD34+ PBPCs from 10 children with solid tumors, we observed no selective loss in CFU-Mk numbers during immunoselection processing and no impairment of clonogenicity. The CFU-Mk yield (59.2 +/- 11.3%) was at least similar to the CD34+ yield (44.2 +/- 3.8%). We assessed the predictive value of CFU-Mk numbers infused for recovery of platelet lineage. We found an inverse correlation between the time taken to reach a platelet count greater than 50 x 10(9)/L and only the CFU-Mk dose (r = -0.71; p = 0.022) among the different type of progenitors, including colony-forming units granulocyte-macrophage (CFU-GM), burst-forming units erythrocyte (BFU-E) and colony-forming units-mixed (CFU-Mix). These findings suggest that CFU-Mk number could be used as sole predictive functional parameter for platelet reconstitution in children after immunoselection of CD34+ cells, in particular for low CD34+ cell dose, and thus as an indicator for initial quality of hematopoietic cells before in vitro expansion.

[Geotrichum capitatum infection in a neutropenic patient. Apropos of a case and review of the literature]. / Infection à Geotrichum capitatum chez un patient neutropénique. A propos d'un cas et revue de la littérature.

INTRODUCTION: Geotrichum capitatum sepsis are rare, occurring exclusively in immunocompromised patients. EXEGESIS: We report the case of a patient with acute leukemia, presenting with chemotherapy-induced neutropenia and hospitalized in an intensive care unit for a severe sepsis. In spite of an antibiotic and antifungal treatment, the patient died of cardiorespiratory failure. Later on, blood cultures proved to be positive for Geotrichum capitatum. CONCLUSION: If fungal infections are common in neutropenic patients, Geotrichum capitatum sepsis remain exceptional. The portal of entry is digestive or respiratory, and the invasion is favored by immunodepression and suppression of the normal microbial flora. Induced lesions can be multiorganic. The treatment is not well established, and the association of either amphotericine B and 5-fluorocytosine or amphotericine B and itraconazole would lead to better results. Nevertheless, the prognosis is still unfavorable, with a mortality rate of approximately 75%.

Bilateral eyelid localisation of a lymphoplasmacytoid lymphoma.

Eyelid localisation of non-Hodgkin's lymphoma is rare, and even more so when it is bilateral. We report a 58 year-old man who presented with an eyelid localisation of lymphoplasmacytoid lymphoma. The initial treatment was chemotherapy with good improvement but the relapse lead us to give radiotherapy with no further relapse 20 months later. Radiotherapy is the current treatment of localised eyelid lymphomas with excellent results. The prognosis is related to the initial staging and the 10-year survival rate is close to 80%.

Autoimmune thrombocytopenia after six cycles of fludarabine phosphate in a patient with chronic lymphocytic leukemia.

Fludarabine phosphate (FDR) has demonstrated a remarkable clinical activity in chronic lymphocytic leukemia (CLL). Myelosuppression is the main toxicity although autoimmune hemolytic anemia (AIHA) is frequently reported. The pathogenesis of AIHA is still unknown however the role of T-cell immunosuppression is suspected. One case of thrombopenia after FDR has been described in a patient with a previous history of an autoimmune thrombocytopenia. We here report a 73-year-old man with a B-CLL and no previous autoimmune disorder who received six courses of fludarabine phosphate and developed afterwards an autoimmune thrombocytopenia.